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The murine seminiferous epithelial cycle is pre-figured in the Sertoli cells of the embryonic testis

Paula M. Timmons^*,, Peter W. J. Rigby and Françoise Poirier

Division of Eukaryotic Molecular Genetics, MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK
^* Present address: Laboratory of Mammalian Neurogenesis, MRC Clinical Sciences Centre, Hammersmith Hospital, Du Cane Road, London W12 0NN, UK
Present address: Section of Gene Function and Regulation, Institute of Cancer Research, Chester Beatty Laboratories, 237 Fulham Road, London SW3 6JB, UK
Present address: Département de Biologie du Développement, Institut Jacques Monod, 2 Place Jussieu, 75005 Paris, France

View larger version (51K): [in a new window]   Fig. 1. Northern analysis of galectin 1 transcripts in postnatal mouse testis. RNA samples from P7, P14, P28 and P56 testes, P56 epididymis and P56 XXSxra testes all contain galectin 1 transcripts migrating as a cluster of bands at approximately 0.6 kb. A discrete band of slower migrating galectin 1 transcripts (arrowhead) is present only in the P28 and P56 testis RNA. Each track contains 10 µg of total RNA.

View larger version (173K): [in a new window]   Fig. 2. Distribution of galectin 1 mRNA in the adult mouse testis. Sections were hybridised with 35S-labelled antisense probes for galectin 1 (A-D,F) or Ctsl (E) and counterstained with Toluidine Blue. (A,B) Brightfield and darkground images showing the different levels of galectin 1 mRNA expressed in individual cross sections of the seminiferous tubules. (C,D) Sections of tubules showing differential expression of galectin 1 mRNA at various stages in the seminiferous epithelial cycle, indicated by Roman numerals in C. Galectin 1 mRNA is most abundant at stages X-XII, and lowest at stages VII-VIII. Transcripts are also detected in interstitial cells lying between the tubules (arrows). (E) mRNA of the Sertoli cell marker Ctsl (dense black grains) is localised around the perimeter of the tubules. (F) Stage VIII-IX tubule (also marked * in A) undergoing spermiation. Black grains indicate expression of galectin 1 mRNA in the Sertoli cells. Arrowheads indicate Sertoli cell nuclei. Autoradiographic exposure times were 14 days for galectin 1 and 12 days for Ctsl.

View larger version (173K): [in a new window]   Fig. 3. Distribution of galectin 1 protein in the adult testis. Sections of Bouin fixed adult mouse testis were stained with Haematoxylin and Eosin (A,C,E) or by anti-galectin 1 alkaline phosphatase immunohistochemistry (B,D,F). (A,B) Galectin 1 immunoreactivity (purple stain) in the wall of a large blood vessel (arrowhead), and interstitial cells (arrows). (C,D) Seminiferous tubules at all stages of the epithelial cycle show strong galectin 1 immunoreactivity in the Sertoli cells, and weaker staining surrounding many germ cell stages. (E,F) Stage VIII tubule with staining of basal and apical regions of the seminiferous epithelium. Groups of round spermatids (asterisks in F) are unstained.

View larger version (171K): [in a new window]   Fig. 4. Galectin 1 expression in the developing testis. In situ hybridisation of testis sections with 35S-labelled antisense probes and Toluidine Blue counterstaining (A,C-N), or anti-galectin 1 alkaline phosphatase immunohistochemistry (B). (A,B) Adjacent serial sections of P7 wild-type testis showing expression of galectin 1 mRNA (A) and protein (B) in testis cords (c), tunica albuginea (t), interstitial cells (arrows), peritubular cells (arrowheads) and blood vessel (v). Note the variable intensity of mRNA and protein staining in the individual testis cord cross sections, and the localisation of signal in the Sertoli cell cytoplasm at the centre of the cords. (C,D) Brightfield and darkground images of P4 testis section probed for Wilm’s tumour (Wt1) mRNA. Signal intensity appears uniform over all testis cords. (E,F) Newborn (P0) testis showing localised differential expression of galectin 1 in the cords. (G,H) E18 testis showing galectin 1 expression in a discrete region of testis cord (arrows). (I,J) E16 testis, showing uniform signal intensity in all testis cords. (K,L) Consecutive serial sections of P8 XXSxra testis, probed for galectin 1 and Amh, respectively. (M,N) Longitudinal stretch of P8 XXSxra testis cord showing junction between regions of low and high galectin 1 mRNA expression. Autoradiographic exposures (in days) were 4 (A,E,F), 9 (C,D), 18 (G-J) and 3 (K-N).

View larger version (160K): [in a new window]   Fig. 5. Coordinated, cyclical gene expression in P8 Sertoli cells. In situ hybridisation on consecutive serial sections of P8 XXSxra testis with probes for Ctsl (A), Sgp2 (B), galectin 1 (C), Amh (E) and Rbpl (F). (D) Diagram of the section in C showing cord segments with relatively high levels of galectin 1 mRNA (filled areas and arrows in C). Corresponding cord segments are indicated by arrows in other panels, showing that the relative signal intensities for galectin 1 and Rbpl are inversely related to the levels of Ctsl, Sgp2 and Amh. Exposure times in days were 3 for Amh, galectin 1 and Sgp2, 6 for Ctsl and 19 for Rbpl.

View larger version (124K): [in a new window]   Fig. 6. Distribution of apoptotic germ cells in P7 testis cords. (A) Section of wild-type P7 testis probed for galectin 1 mRNA by in situ hybridisation. (B) Adjacent serial section labelled by TUNEL and counterstained with Methyl Green. Apoptotic bodies appear as brown spots. (C) Next serial section stained by galectin 1 immunohistochemistry. A group of testis cords is shown to the right of each panel at a higher magnification to illustrate the high (H), medium (M) and low (L) categories of signal intensity. Segments of testis cord that contain apoptotic germ cells (labelled by TUNEL in B) are outlined in each panel.